Theses/Dissertations - Biomedical Studieshttp://hdl.handle.net/2104/4800
Thu, 12 Feb 2015 00:59:13 GMT2015-02-12T00:59:13ZAdvancing chemical and biologically-based “Read across” through in silico, in vitro and in vivo techniques.http://hdl.handle.net/2104/9148
Advancing chemical and biologically-based “Read across” through in silico, in vitro and in vivo techniques.
Connors, Kristin A.
Read across approaches include data leveraging across chemicals or among biological scales of organization or species, and may focus limited resources to support chemical hazard and risk assessment. Sustainable molecular design of less toxic industrial chemicals promises to preempt the production of organic contaminants with elevated toxicity. In chapter 2, probabilistic hazard assessment approaches were used to model potential reduction of aquatic toxicity hazards by using the ‘rule of two’ in the chemical design stage. Implementing this 'rule of two' was predicted to appreciably decrease chemicals designated of 'high' and 'very high' concern for standardized acute and chronic toxicity in common aquatic species. However, select modes of action and chemical classes will not have a reduced toxicity profile after following the 'rule of two.' Other read across techniques were examined to support chemical prioritization for safety assessments or to generate hypotheses for advanced research. Unlike most industrial chemicals, robust data exists for pharmaceutical physicochemical and biological properties, including human metabolism. Thus, it may be possible to employ existing mammalian pharmacological safety data to support screening-level bioaccumulation assessments. In chapter 3, a comparative pharmacology approach was used to determine whether rainbow trout biotransform pharmaceuticals known to be substrates for specific human CYPs. Only two general CYP substrates demonstrated measureable intrinsic clearance in vitro. No significant biotranformation was observed for substrates of human CYP2D6, CYP2C9, or CYP3A4. In chapter 4, enantiomer specific biotransformation profiles of chiral pharmaceuticals also deviated from human clearance rates. These observations demonstrate that relative clearance rates in trout are not predictable from human pharmacology data. Biological read across extrapolations were further explored in chapter 5 to characterize hazards of next generation therapeutics in zebrafish. Mechanistic and behavioral responses identified potential adverse outcomes from emerging antidepressants.
Fri, 05 Sep 2014 00:00:00 GMThttp://hdl.handle.net/2104/91482014-09-05T00:00:00ZTargeting the immune microenvironment to treat breast cancer.http://hdl.handle.net/2104/9117
Targeting the immune microenvironment to treat breast cancer.
Xu, Kangling.
In tumor microenvironment, the crosstalk between infiltrating inflammatory cells and tumor cells creates a cytokine milieu which can promote both oncogenesis and tumor rejection. We have found an IL-13 dominant cytokine environment existing in breast cancer tissue, and the IL-13 is pathogenic since blocking IL-13 using a neutralizing antibody in vivo could prevent tumor growth. Furthermore, DCs are involved critically in breast tumor progression, very possibly through regulating Th1/Th2 polarization, since our previously in vivo data show that in the absence of DCs, CD4⁺ and CD8⁺ T cells fails to facilitate tumor growth in a xenograft model. Thus, it is important to unveil how DCs behave in the breast cancer microenvironment, and how DCs crosstalk with cancer cells or other stromal cells. In this study, we have shown that pro-tumor inflammation in breast cancer is driven by cancer cell-derived thymic stromal lymphopoietin (TSLP) that induces OX40L expression on dendritic cells (DCs). OX40L⁺ DCs generate inflammatory CD4⁺ T cells producing TNF-α and IL-13 (iTh2). Furthermore, we found high levels of IL-1β in breast cancer microenvironment of the patients. IL-1β level is positively correlated with the levels of IL-13 and TSLP. Importantly, IL-1β level is associated with the stage of the disease. IL-1β induces TSLP production from breast cancer cells lines in a dose- dependent manner in vitro. Cancer cells induce IL-1β both transcriptionally and translationally in mDCs and monocytes in a contact-dependent manner. This is mediated by cancer cell-derived TGF-β. Moreover, TGF-β -activating kinase 1 (TAK1) signaling is involved in caspase-1 activation and TGF-β-dependent IL-1β production. Blocking TGF-β in vivo prevents tumor growth through decrease IL-1β production. Administration of IL-1R antagonist anakinra prevents tumor growth in vivo, blocks OX40L expression on mDCs, and blocks iTh2 generation. Thus targeting the immune microenvironment through blockade of IL-1β represents a novel approach to treat breast cancer.
Wed, 11 Jun 2014 00:00:00 GMThttp://hdl.handle.net/2104/91172014-06-11T00:00:00ZReprogramming the immune environment in breast cancer via dendritic cells.http://hdl.handle.net/2104/8864
Reprogramming the immune environment in breast cancer via dendritic cells.
Wu, Te-Chia.
Breast cancer is the most common cancer among American women. The current therapies are not curative for some forms of breast cancer, especially breast cancers that do not express the growth factor and hormone receptors and metastatic cancers. Recently, immunotherapy become an anticipated option for breast cancer. Before approaching immunotherapy, it is important to firstly understand how is the immune microenvironment regulated in breast cancer to determine how it could be modulated for therapy.
The human breast cancer microenvironment displays features of T helper 2 (Th2) immunity which promotes tumor development. Here we show that human breast cancer cells produce thymic stromal lymphopoietin (TSLP). Breast tumor supernatants, in a manner dependent on TSLP, induce expression of OX40L on dendritic cells (DCs). OX40L⁺ DCs are found in primary breast tumor infiltrates. OX40L⁺ DCs drive development of inflammatory Th2 cells producing interleukin 13 and tumor necrosis factor in vitro. Antibodies neutralizing TSLP or OX40L inhibit breast tumor growth and interleukin 13 production in a humanized mouse model of breast cancer. Thus, breast
cancer cell-derived TSLP, by inducing OX40L expression on DCs, contributes to the Th2 immunity conducive to breast tumor development.
In order to reprogram the inflammatory pro-tumor Th2 (iTh2) into anti-tumor Th1 microenvironment, we tested the impact of targeting the innate receptors on DCs to render the resistant to tumor environment. We show that intratumoral delivery of β-glucan, a natural ligand for dectin-1 expressed on DCs, blocks the generation of iTh2 cells leading to decreased IL-13 in the tumor microenvironment and prevents breast cancer development in vivo. β-glucan inhibits OX40L expression on tumor-associated DCs which is due to a block in STAT6 phosphorylation. β-glucan-treated DCs, when exposed to breast cancer supernatant, secrete higher levels of IL-12p70 and do not expand iTh2 cells thereby enabling the T helper 1 cells secreting IFN-γ. β-glucan exposed DCs expand CD8⁺ T cells that express CD103, a ligand for E-cadherin. These CD8+ T cells, which produce higher IFN-γ, Granzyme A and Granzyme B, accumulate in the tumors leading to enhanced tumor necrosis in vivo. DC reprogramming by β-glucan is dependent upon dectin-1 engagement. The ligand of TLR7/8 (CL075) and TLR3 (polyI:C) show the same effect in Th2 response inhibition but no effect on the functions of CD8 T cells. Taken together, our data demonstrate that exploiting pattern recognition receptors on tumor-infiltrating DCs enables cancer rejection.
Tue, 24 Sep 2013 00:00:00 GMThttp://hdl.handle.net/2104/88642013-09-24T00:00:00ZDevelopment of an aptamer-based detection assay against Bacillus anthracis lethal factor and Escherichia coli.http://hdl.handle.net/2104/8838
Development of an aptamer-based detection assay against Bacillus anthracis lethal factor and Escherichia coli.
Lahousse, Mieke.
The impressive capacity of bacteria to adapt to their environment has led to the development of mechanisms that can convey resistance to currently FDA approved antibiotics. In an attempt to find a solution to the possibility that soon there will be no antibiotic treatment available for bacterial infections, new approaches are being explored. One such approach relies on the effective detection and/or diagnosis of pathogens or unique bacterial proteins to aid in prevention or better treatment of infectious diseases. Within this work we describe the development of ssDNA aptamers by SELEX that can bind to their target with high affinity and can be used to develop colorimetric assays that can positively detect the presence of the target. Our targets for aptamer development were whole E. coli cells, which are currently used in water quality assessment, and a unique protein toxin (lethal factor) produced by B. anthracis, the etiological agent of anthrax. As a result of the SELEX experiments we recovered one aptamer that binds to LF protein with high affinity and we demonstrated that its use in the development of an aptamer-based colorimetric assay for LF was successful. We also demonstrate that the aptamer found for LF protein can act as an inhibitor of the catalytic activity of LF, suggests its potential use as a therapeutic agent. The SELEX experiment using whole E. coli cells generated a diverse number of aptamers with binding affinities in the low µM range. Additionally, studies of aptamer specificity showed that three aptamers had no binding affinity for other coliform strains, making them suitable aptamers for further characterization. This work also describes the characterization of a glutaredoxin protein expressed by Synechocystis sp. involved in arsenate reduction. Elucidation of the mechanism by which cyanobacteria can survive in highly arsenic contaminated environments could enlighten possible remediation approaches. In conclusion, we successfully developed ssDNA aptamers that can bind with high affinity and specificity to its targets with demonstrated potential as detecting agents and contributed to the enrichment of the knowledge available for glutaredoxin GrxA involved in arsenate oxidoreduction by Synechocystis sp.
Tue, 24 Sep 2013 00:00:00 GMThttp://hdl.handle.net/2104/88382013-09-24T00:00:00Z